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STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF THE OLD FAMILY NUCLEASES

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Abstract

Overcoming lysogenization defect (OLD) proteins were first discovered in bacteriophage P2, where presence of the old gene plays a role in restricting co-infection of lambda phage in the E. coli host. These enzymes are well conserved and present in a wide array of bacteria, archaea, and viruses, yet their function and mechanism in other contexts remain a mystery. Structurally, these enzymes contain an N-terminal ATPase domain and a C-terminal Toprim domain common amongst topoisomerases, DnaG primases, gyrases, RecR recombination proteins, and 5S rRNA maturases. The Toprim-containing C-teriminal region of the OLD proteins is sufficient for nuclease activity and acts on both circular and linear DNA. Crystal structures of the C-terminal region of OLD proteins from Burhkolderia pseudomallei and Xanthomonas campestris pv campestris revealed that OLD proteins possess a topologically unique Toprim domain and utilize a two-metal catalytic mechanism to hydrolyze DNA. In order to understand the role of the N-terminal ATPase domain and the overall architecture of OLD proteins, I purified and solved the crystal structure of a Class 1 full-length OLD protein from Thermus scotoductus (Ts). The ATPase domain of TsOLD is topologically similar to the ABC-type ATPases, whose members include the ABC transporters, SMC proteins, and the DNA repair protein Rad50. The structure highlights unique active site resides used to bind and hydrolyze ATP that distinguish the OLD proteins from other ABC ATPases. Together these data provide novel insights into the mechanism and function of OLD family nucleases.

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2019-05-30

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Biochemistry

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Chappie, Joshua S.

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Helmann, John D.
Weiss, Robert S.
Ke, Ailong

Degree Discipline

Biochemistry, Molecular and Cell Biology

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Ph.D., Biochemistry, Molecular and Cell Biology

Degree Level

Doctor of Philosophy

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Government Document

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dissertation or thesis

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